The present invention relates to competitive protein binding assay, and more particularly, to competitive protein binding assay for various low molecular weight ligands such as digoxin, T.sub.3 uptake, thyroxine, cortisol, insulin and for simultaneous determination of folate and vitamin B.sub.12.
Generally, it is well known to measure endogenous low molecular weight ligands such as folate or vitamin B.sub.12 by a competitive protein-binding technique. For example, competitive protein-binding for the assay of either folate or vitamin B.sub.12 involves the ability of unlabled folate or vitamin B.sub.12 in serum or other media to compete with a known quantity of labeled folic acid or labeled vitamin B.sub.12 for specific sites on a folate protein binder or vitamin B.sub.12 protein binder and thereby inhibit the binding of the labeled folic acid or vitamin B.sub.12. As a result of the competitive inhibition, the ratio of bound labeled folic acid or vitamin B.sub.12 to free labeled folic acid or vitamin B.sub.12 diminishes as the concentration of unlabeled folate or vitamin B.sub.12 is increased. Accordingly, the concentration of folate or vitamin B.sub.12 in an unknown sample, such as a patient's serum, is obtained by comparing the inhibition (ratio of bound labeled folic acid or vitamin B.sub.12 to free labeled folic acid or vitamin B.sub.12) observed with that produced by known amounts of folate or vitamin B.sub.12, as presented in a standard curve. Such measurements generally require successive runs for the determination of folate and of vitamin B.sub.12.
U.S. Pat. No. 4,146,602 to Gutcho et al. describes a method for the simultaneous radioassay of folate and vitamin B.sub.12. In the method of the Gutcho patent, a sample containing folate and vitamin B.sub.12, free of endogenous binder therefor, is contacted with a protein binder for the folate, a separate protein binder for the vitamin B.sub.12, a folate tracer labeled with radioiodine and a vitamin B.sub.12 tracer labeled with radiocobalt. The bound portions of folate and vitamin B.sub.12 are then separated from the free portions of folate and vitamin B.sub.12. The radioactivity of at least one of the bound and free portions is then counted and compared to standard curves to determine both the folate concentration and the vitamin B.sub.12 concentration. Of course, separate counts are required to determine radioiodine and radiocobalt.
As described in the Gutcho et al. patent the bound and free portions of the folate and vitamin B.sub.12 are separated by procedures known in the art. Such procedures, as described in the Gutcho et al. patent generally rely on the use of a particulate adsorbent. The sample containing the bound and free portions of labeled and unlabeled folate and vitamin B.sub.12 are mixed with the particulate adsorbent. The mixture of adsorbent and sample is maintained at room temperature, usually under turbulent conditions, for a period of time to permit contact of the labeled folate and vitamin B.sub.12 with the particulate adsorbent. The mixture of sample and particulate adsorbent is then centrifuged for a period of time, usually about 15 minutes, and the sample is separated into bound and unbound portions by decanting the unbound portion from the bound portion retained by the adsorbent.
The Gutcho et al. patent indicates that various adsorbents, such as ion exchange resins and inorganic adsorbents can be used to separate the bound and unbound portions. The only adsorbent mentioned, however, is dextran coated charcoal. Such reference to a variety of useful adsorbents, moreover, is in the context of the separation method used in the Gutcho et al. patent, i.e., a batch separation requiring a lengthy contact period and centrifugation.
It would be desirable to adapt a competitive protain binding assay, such as a simultaneous B.sub.12 /folate assay system to the automated methods available for separating bound and unbound fractions. Such automated systems utilize a continuous stream of a treated sample which is passed through a column containing a separation medium capable of removing the unbound fraction from the sample by antibody/antigen coupling, adsorption or other physical means. Such systems are usually provided with automatic counters capable of counting both radioiodine and radiocobalt and also capable of monitoring both the free and bound fraction through successive use of a coupling buffer and an elution buffer. An integrated program is then used to provide a direct readout in terms of percent bound. The percent which is bound to the separation medium in the column is then related to standard curves to provide a measure of the concentration of folate, vitamin B.sub.12 or other analyte present in the sample.
To be useful in a radioassay system, the binding material must have a high separation efficiency (maximum binding of free fraction) as well as stability and the ability to be eluted for counting of the bound fraction and recovery of the resin for future runs. In respect to competitive protein binding assays, the separation medium must also have a low affinity for the protein in the sample matrix and the protein used to effect competitive binding of labeled and unlabeled analyte. Surprisingly, the separation media most extensively used in competitive protein binding radioassay systems have not been found to be suitable for the simultaneous assay of folate and vitamin B.sub.12. The dextran coated charcoal described in the Gutcho et al. patent was found to work well in an aqueous environment but provided poor maximum binding with the sample in a protein matrix, such as serum.